Reviewed & Approved by: Dr. Guangming Xiong, Head of the Pharmacology and Toxicology, University Kiel, Germany.

Abstract

Phenoxyl radicals can covalently attach to the C8-site of 2′-deoxyguanosine (dG) to generate oxygen-linked biaryl ether C8-dG adducts. To determine the mutagenicity of an O-linked C8- dG adduct, C8-phenoxy-dG (PhOdG) was incorporated into the G3 position (X) of the NarI recognition sequence within a 22-mer oligonucleotide template (5′-CTCGGCX-CCATCCCTTACGAGC, where X = dG, or PhOdG) using solid-phase DNA synthesis. The NarI(22) template was annealed to a 15-mer primer and in vitro mutagenicity was assessed using primer-extension assays with a high-fidelity replicative polymerase, Escherichia coli pol I Klenow fragment exo− (Kf−), and a lesion-bypass Y-family polymerase, Sulfolobus solfataricus P2 DNA polymerase IV (Dpo4). These studies predict that the O-linked C8-dG lesion PhOdG will have a low mutagenic effect, and is unlikely to contribute strongly to phenol toxicity.

Our interest in phenol toxicity stems from the finding that certain phenolic radicals can attach covalently to the C8-site of 2′-deoxyguanosine (dG) to afford C8-dG adducts [11-14]. Due to the ambident electrophilicity of phenoxyl radicals (C vs O) both carbon-and oxygen-linked C8-dG adducts have been observed (Figure 1). The phenolic C-linked C8-dG adducts belong to a larger class of C8-aryl-dG lesions produced by a number of chemical mutagens that include polycyclic aromatic hydrocarbons (PAHs) [15], estrogens [16], nitroaromatics [17] and arylhydrazines [18]. The phenolic O-linked C8-dG adducts are unique to phenol precursors, but are expected to be structurally similar to the corresponding nitrogen-linked C8- dG adducts produced by aromatic amine carcinogens [19-21]. For aromatic amines, structure-activity relationships have demonstrated that all of the potent carcinogens are polycyclic structures, while none of the single-ringed aniline derivatives are potent carcinogens [22].

Figure 1: Ambident reactivity of phenoxyl radical at the C8-site of dG.

We recently incorporated C8-phenoxy-2′-deoxyguanine (PhOdG) into the G3 position of the NarI recognition sequence within a 12-mer oligonucleotide (5′-CTCGGCXCCATC, G3 = X = PhOdG) in order to study its structural characteristics to predict mutagenic outcome [23]. The NarI sequence is a hot-spot for frameshift mutations mediated by the N-linked C8-dG adduct of N-acetyl- 2-aminofluorene (AAF) [19-21]. Within the NarI(12) duplex, the PhOdG lesion was shown to adopt the major groove B conformation opposite C, with minimal disruption to the duplex structure [23]. These findings correlated with the conformational preference for the corresponding single-ringed N-linked C8-dG adduct produced by aniline [24]. On the basis of this comparison, the PhOdG lesion was predicted to be weakly mutagenic [23].

In the present study we utilize primer-extension assays to more accurately predict the mutagenic potential of the O-linked PhOdG adduct. In our model the PhOdG adduct has been incorporated into the G3 position of a NarI(22) template (5′-CTCGGCXCCAT-CCCTTACGAGC, where X = dG, or PhOdG) and annealed to a 15- mer primer strand (5′-GCTCGTAAGGGATGG). The bypass ability in vitro of the high-fidelity replicative DNA polymerase – E. coli DNA polymerase I Klenow fragment (exonuclease negative mutant, Kf−) was compared to bypass by the Y-family DNA polymerase IV (Dpo4) from Sulfolobus solfataricus [25]. High-fidelity polymerases utilize an ‘induced-fit’ mechanism of replication, and bulky adducts in the template strand often stall or block the progression of replication through distortion of the polymerase active site [26]. Such stalling in vivo is believed to be a trigger for the recruitment of Y-family translesion polymerases [26], which have a larger solvent-exposed active site to accommodate bulky DNA lesions for more probable effective bypass [27]. Mutagenesis experiments in vitro support our predictions derived from structural analysis of the PhOdG lesion within NarI(12) [23], and suggest that the O-linked C8-dG adduct is unlikely to contribute strongly to phenol toxicity.

Materials and Methods

Materials

The NarI(22) oligonucleotide substrates were prepared on a BioAutomation MerMade 12 automatic DNA synthesizer using standard or modified β-cyanoethylphosphoramidite chemistry (1 μmol scale). Full synthetic details of the PhOdG phosphoramidite have been previously published [23]. The 15-mer primer oligonucleotide was purchased from Sigma Genosys (Oakville, ON, Canada) and was purified by Sigma using polyacrylamide gel electrophoresis (PAGE). The purity of the oligonucleotide sample was checked at the University of Guelph using HPLC (see Supporting Information for HPLC analysis of the 15-mer primer (Figure S1)). Escherichia coli pol I Klenow fragment exo− (Kf−) and T4 polynucleotide kinase were purchased from New England BioLabs, while Sulfolobus solfataricus P2 DNA polymerase IV (Dpo4) was purchased from Trevigen Inc; isotopically-labeled ATP ([γ-32P]-ATP) was purchased from PerkinElmer.

Stock solutions of DNA (0.50 mL or 1.00 mL) were prepared in purified water. Consecutive additions of 5 μL of DNA stock to 1985 μL of purified water followed by an absorption scan at 260 nm on a Cary 300-Bio UV-Visible spectrophotometer equipped with a Peltier block-heating unit and automated temperature controller using standard 10 mm light path quartz glass cells from Hellma GmbH & Co (Concord, ON). Scans were performed three times in order to determine the concentration of the stock solution. Molar absorptivities (ε) of the unmodified NarI(22) were used for the modified NarI(22) strand and were calculated online using Integrated DNA Technologies (IDT) OligoAnalyzer 3.1.

T4 polynucleotide kinase and [γ-32P] ATP were used to label the 15mer primer strands at the 5′-end. The unmodified and modified DNA template: primer duplexes were prepared by annealing the 15mer primer and NarI(22) template strands (50% excess of template strand) through heating the mixtures to 95ºC for ten minutes, followed by slow cooling to room temperature overnight.

Single nucleotide incorporation assays

Kf− or Dpo4 were used to perform primer extension reactions on each previously labeled and annealed template: Primer duplex in the presence of 25 μM of dCTP, dGTP, dATP, or dTTP. Reactions were initiated by the addition of the dNTP to enzyme/DNA mixtures to give a final reaction volume of 10 μL. The final concentrations for Kf− assays were 50 mM NaCl, 10mM Tris-HCl (pH 7.9), 10mM MgCl2, 1mM dithiothreitol (DTT), 100 nM duplex, and 20 nM Kf−. The final concentrations for Dpo4 assays were 50 mM NaCl, 50 mM Tris (pH 8.0), 2.5 mM MgCl2, 5 mM DTT, 100 μg/mL bovine serum albumin (BSA), 5% glycerol, 100 nM duplex, and 20 nM Dpo4. Reactions were incubated at 37ºC for 1 hour with Kf−, and 30 minutes with Dpo4, followed by 4 μL transferred and mixed with 36 μL of loading dye (95% formamide, 20 mM EDTA, 0.05% xylene cyanol and bromophenol blue) to terminate the reaction. 4 μL of these quenched reactions were then subjected to 15% polyacrylamide gel electrophoresis in the presence of 7 M urea and incorporation products were visualized using a Bio-Rad phosphorimager. Relative frequency of base incorporation was determined using ImageJ64 software.

Full length extension assays

Kf− or Dpo4 were used to perform primer extension reactions on each previously labeled and annealed template:primer duplex in the presence of a 100 μM 4dNTP mix (25 μM each dNTP). A final concentration of 20 nM of Kf− or Dpo4 was used in each reaction, while all other final concentrations were the same as outlined for the single nucleotide incorporation assays. Reactions were initiated by the addition of the 4dNTP mix to enzyme/DNA mixtures to give a final reaction volume of 10 μL with Kf−, or 20 μL with Dpo4. Reactions with Kf− were incubated at 37ºC for 1 hour, and then quenched in the same manner as described above. Reactions with Dpo4 were also incubated at 37ºC, but 4 μL aliquots were removed from incubation after 2, 5, 10, 15, and 30 minutes with the unmodified duplex, and after 15, 30, 45, 60, and 90 minutes with the modified duplex. Each 4 μL aliquot removed was quenched in the same manner as listed above. All quenched reaction samples were then subjected to 15% polyacrylamide gel electrophoresis in the presence of 7 M urea and incorporation products were visualized using a Bio-Rad phosphorimager.

Results and Discussion

Primer extension by Klenow fragment exo− (Kf−) Single nucleotide insertion assays using Kf− were carried out using the NarI(22):15mer template:primer (Figure 2). On the unmodified NarI(22) template (X = dG), Kf− mainly incorporated the correct base C (~ 82%, Figure 2b). At 20 nM enzyme concentration, significant amounts of G (~37%) and T (~30%) were also incorporated opposite dG during the 60 min reaction time. On the modified NarI(22) template (X = PhOdG), the polymerase again mainly inserted the correct base C (~70%, Figure 2b). However, a higher relative frequency of a second C (10%) and a slight increase in the insertion of A compared to the unmodified template was observed. The PhOdG adduct also inhibited misincorporation of G and T compared to the unmodified template (Figure 2b).

Figure 2: a) Single nucleotide incorporation primer extension assays with 25μM of individual dNTPs as indicated under each lane, by incubation of 20 nM of Kf− with each duplex (sequence presented above gel) for 1 hour at 37°C. (Note: B = blank where no enzyme present). b) Relative frequency of each dNTP incorporated by Kf− on NarI(22):15-mer template:primer duplex where X=dG (black), or X=PhOdG (grey).

In the presence of all four dNTPs (Figure 3), extension past PhOdG was stalled after the incorporation of one nucleotide (presumably C across from PhOdG), though the full length extension product was clearly obtained. The observed stalling at position 1 (numbering on gel in Figure 3) is in agreement with previous reports that extension past bulky C8-dG adducts is more difficult than base insertion opposite the lesion [28]. An 8th band for one base additional extension beyond the template strand was also observed, which is a typical non-template-dependent extension from a blunt end [29].

Figure 3: a) Full length primer extension assays on NarI(22):15-mer template:primer duplexes (sequence presented above gel) where X=dG or PhOdG by 20 nM Kf− in the presence of 25 μM of each dNTP. Reactions were incubated for 60 min at 37°C. The blank sample had no enzyme present during the reaction with unmodified duplex.

Translesion synthesis by Dpo4

Given that the PhOdG adduct stalls replication by the high-fidelity polymerase Kf−, it was desirable to examine extension using a lesion-bypass DNA polymerase. Distortions and attendant stalling of high-fidelity polymerases by bulky adducts are regarded as signals for recruiting lesion-bypass polymerases in vivo [26]. Thus, bulky phenolic adducts are more likely to be processed by translesion polymerases and to model this interaction we employed the Y-family polymerase, Sulfolobus solfataricus P2 DNA polymerase IV (Dpo4) [25,26].

Single nucleotide insertion assays using Dpo4 were carried out using the NarI(22):15mer template:primer (Figure 4). On both the unmodified and modified NarI(22) templated, Dpo4 exhibits low-fidelity with significant amounts of misincorporations (Figure 4b). For unmodified templates, this phenomenon is known for translesion polymerases due to decreased interactions of the larger, more spacious active site with the template DNA, providing lower geometric selection for correct base pairs [30]. Typically, the Y-family polymerases will exhibit much higher fidelity when replicating damaged DNA [31]. However, in this instance the PhOdG adduct had little impact on the fidelity of Dpo4 compared to the unmodified control (Figure 4b).

Figure 4: a) a) Single nucleotide incorporation primer extension assays with 25μM of individual dNTPs as indicated under each lane, by incubation of 20 nM of Dpo4 with each duplex (sequence presented above gel) for 30 min at 37°C. (Note: B = blank where no enzyme present). b) Relative frequency of each dNTP incorporated by Dpo4 on NarI(22):15-mer template:primer duplex where X=dG (black), or X=PhOdG (grey).

In the presence of all four dNTPs (Figure 5), the unmodified template was fully extended to the 22-mer complementary strand by 5 min of incubation with Dpo4, with some additional incorporation of an 8th base for one base additional extension from a blunt end [29].

Figure 5: a) Full length primer extension assays on NarI(22):15-mer template: primer duplexes (sequence presented above gel) where X=dG or PhOdG by 20 nM Dpo4 in the presence of 25 μM of each dNTP. Aliquotsof each reaction were removed from incubation at 37°C after the time listed under each lane. (Note: B = blank where no enzyme present).

Conclusions

The current study has allowed us to conclude the following: (1) the single-ringed oxygen-linked C8-phenoxy-dG adduct (PhOdG) does not strongly impede the progress of DNA replication by either Kf− or Dpo4 when inserted into the NarI(22) template and annealed to a 15-mer primer. Both the high-fidelity polymerase Kf− and the lesion-bypass polymerase Dpo4 are able to fully extend the 15-mer primer in the presence of the PhOdG lesion, although PhOdG causes some stalling after the first incorporation opposite the adduct. (2) In single nucleotide insertion assays, the PhOdG adduct does not strongly alter the relative frequency of dNTP incorporation compared to insertion opposite dG. Overall, these results suggest that PhOdG is a weakly mutagenic lesion, which correlates with our earlier prediction for PhOdG based on its structural characteristics within the NarI(12) duplex. These findings suggest that the O-linked PhOdG adduct will not strongly contribute to phenol toxicity. Our results are the first to report the in vitro mutagenicity of an oxygen-linked biaryl ether C8-dG adduct and provide a basis for comparison to other O-linked C8-dG adducts derived from phenolic toxins.